The long range goal of this work is to define how bacterial cell division is regulated. Our approach has been to isolate and study conditional cell division mutants which are defective at high temperature (ts) specifically in septum formation. These ts mutants, which grow as long, non-septate filaments at high temperature, are defective in the sep and ftsA genes. sep and ftsA are located near leuA in a cluster of genes involved in membrane-wall biosynthesis or function. One model to explain the regulation of cell division predicts that the expression of specific cell division genes (such as sep and ftsA) is controlled in a cyclic manner during the cell cycle. sep has been cloned in a viable phage vector. This hybrid phage and direct assays of sep mutants were used to demonstrate that the sep product is a penicillin-binding protein (PBP-3).